ABSTRACT Multiple sclerosis (MS) is a chronic disease of the central nervous system that causes cognitive and physical disability. MS is characterized by demyelination, inflammation, lesion formation and neurodegeneration. Cholesterol is a key lipid in the myelin sheath and is critical for myelin function. Disease progression is a significant challenge in MS therapeutics because the currently available immunomodulatory drugs do not halt the neurodegenerative processes that are responsible for brain atrophy and disability progression. Compelling recent evidence from our research indicates that serum cholesterol and lipoprotein profiles are associated with disability changes and magnetic resonance imaging (MRI) measures of lesion formation and neurodegeneration in patients with established MS and in the earliest stages of MS following the first demyelinating event. The mechanisms underlying these pathophysiological associations of cholesterol biomarkers with brain injury in MS are not known. Based on our preliminary results, we hypothesize that the associations between serum cholesterol and brain injury in MS are driven by key contributions from oxysterols, which are promising molecular mediators for these pathophysiological associations because they can cross the blood brain barrier and also regulate cholesterol homeostasis in the brain and the periphery. Oxysterols, particularly 24-hydroxy cholesterol (24HC), 27-hydroxy cholesterol (27HC), 7?-hydroxy cholesterol (7?HC) and 7-ketocholesterol (7KC) are of interest because they are potent transcription factor ligands and regulatory mediators of cholesterol and immune homeostasis. The goal is to investigate the role of oxysterols and cholesterol pathway biomarkers in MS disease progression. In Specific Aim 1, we will compare the longitudinal changes in a focused panel of major oxysterols in serum at baseline and after 5 years in MS patients compared to controls and investigate their associations with serum lipids. In Specific Aim 2, we will measure select downstream molecular targets of oxysterol signaling and identify the oxysterols and oxysterol target molecules associated with progression of brain injury over 5 years. We hypothesize that decreased 24-hydroxy cholesterol, decreased 7?-hydroxy cholesterol, increased 27- hydroxy cholesterol and increased 7-ketocholesterol levels in serum are associated with greater neurodegeneration. The study will use serum samples obtained at baseline and 5-year follow-up from 150 MS patients and 50 controls. The patients' serum samples will be analyzed for oxysterol and oxysterol targets. Aim 1 will leverage sensitive and selective state-of-the-art liquid chromatography-mass spectrometry assays capable of simultaneously quantitating multiple oxysterols. We will have longitudinal clinical and quantitative MRI data for this cohort. When successfully completed, this project will identify molecular mechanisms that will better define the pathophysiological role of the cholesterol pathway in MS disease progression.